Expression and Identification of Mutated Osteoprotegerin in Culture Cells and Larvae of Silkworm

Hai-Long XIAO, Yong-Feng
JIN, and Yao-Zhou ZHANG1*
( Institute of Biochemistry, College of Life Sciences, Zhejiang University,
Hangzhou 310029, China; 1College of Life Sciences, Zhejiang
University of Science & Technology, Hangzhou 310018, China )

Abstract The mutated osteoprotegerin
(OPG-372) gene was inserted into the baculovirus transfer vector pBacPAK8, and
the recombinant plasmid was co-transfected with linearized Bm-BacPAK6 virus
DNA into BmN cells, then homologous recombination occurred inside the cells.
The recombinant virus BmNPV-OPG-372 was screened and identified by Southern
blotting. The recombinant human OPG-372 was expressed in cultured cells and
the larvae of silkworm by inoculation of recombinant virus. The expression products
were run on the SDS-PAGE and their immunoreactivities were determined by Western
blotting. It was found that a 42 kD recombinant protein was expressed in BmN
cells and a 46 kD one in larvae respectively. The bioactivities of the recombinant
proteins were determined by hypocalcemic effect assay in the mice. The results
showed that the recombinant proteins had a significant hypocalcemic effect on
mice sera.

Key words OPG-372; silkworm
baculovirus expression system; hypocalcemic effect

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Received: January 7, 2004 Accepted:
March 31, 2004
*Corresponding author: Tel, 86-571-86843190; Fax, 86-571-86843198; E-mail, [email protected]